Influence of non-MHC genes on lymphocyte response to Mycobacterium tuberculosis antigens and tuberculin status in Pulmonary tuberculosis

Background & objectives : Major histocompatibility complex (MHC) genes are known to influence the immune functions. In the present study, the influence of non-MHC genes such as mannose binding protein (MBP), vitamin D receptor (VDR) and interleukin-1 receptor antagonist (IL-IRA) gene polymorphisms on lymphocyte response to Mycobacterium tuberculosis culture filtrate antigen (10 μg/ml) was studied in 44 patients with active pulmonary TB and the family contacts (35) and in 32 normal healthy subjects. The influence of these gene polymorphisms on tuberculin (1TU of PPD of M. tuberculosis) reactivity status in 146 pulmonary TB patients was also studied. Methods : The MBP and VDR genes were amplified using polymerase chain reaction (PCR) and genotyping was carried out using sequence specific oligonucleotide probes by dot blot and IL-1RA by agarose gel electrophoresis. Results : A significantly decreased lymphocyte response to M. tuberculosis antigen was seen in pulmonary TB patients positive for functional mutant homozygotes of MBP (00) compared to heterozygote carriers (AO; P<0.02) and wild homozygotes (AA; P<0.01). The variant mutant genotype (tt) of VDR gene was associated with an increased lymphocyte response in control subjects compared to active TB patients with tt genotype (P<0.05). Heterozygote carriers of MBP (AO) were associated with a significantly (P<0.001) decreased tuberculin reactivity compared to wild homozygotes (AA). The VDR genotype Tt (heterozygote carrier) was associated with an increased tuberculin reactivity in female TB patients as compared to male patients (P<0.001). Interpretation & conclusions : The present study suggested that MBP and VDR genes influence the cell mediated immune response in pulmonary TB patients. Non-MHC genes along with HLA-Class II genes/gene products may be playing an immunoregulatory role in the mechanism of susceptibility/resistance to tuberculosis

HLA-DR2 subtypes & immune responses in pulmonary tuberculosis

Background & objectives: HLA-DR2 has been shown to be associated with the susceptibility to pulmonary tuberculosis and altered antibody and lymphocyte response in pulmonary tuberculosis. In the present study, the influence of DR2 subtypes on antibody titre and lymphocyte response ,to Mycobacterium tuberculosis culture filtrate antigens (10 μg/ml) was studied in 22 patients with active pulmonary TB (ATB), 50 inactive (cured) TB (ITB) patients and 36 healthy control subjects. Methods. HLA-DR2 gene was amplified by polymerase chain reaction (PCR) and dot-blotted. Genotyping of DRBl*1501, *1502, *1503, *1601 and *1602 was carried out using sequence specific oligonucleotide probes (SSOPs) and detected by chemiluminescence method. Antibody titre as well as lymphocyte response to M.tuberculosis antigens were measured by enzyme linked immunosorbent assay (ELISA) and lymphocyte transformation test (LTT) respectively. Results: The allele frequency of DRB1*15Ol was significantly increased in pulmonary tuberculosis patients as compared to controls (P<0.05). No marked difference in the antibody titre and lymphocyte response to M. tuberculosis antigens was observed between the DRBl *1501, *1502 and *1503 positive or negative controls, ATB and ITB patients. DRBl *1501 and *1502 positive as well as negative ATB patients showed a higher antibody titre as compared to controls and ITB patients. ITB patients with *1502 showed a higher lymphocyte response as compared to *1502 positive controls (P<0.001) and ATB patients (P<0.05). Similarly, an increased lymphocyte response was observed in *1501, and *I503 negative ITB patients compared to *1501 and *1503 negative controls and ATB patients. Interpretation & conclusion: The present study revealed that DRBl *1501 may be associated either alone or with other DR2 alleles, with the susceptibility to pulmonary tuberculosis. None of the DR2 alleles influenced the antibody and lymphocyte response to M tuberculosis culture filtrate antigens. This suggested that HLA-DR2 gene/gene products as a whole may influence the immune response in pulmonary tuberculosis

Influence of HLA-DR2 phenotype on humoral immunity & lymphocyte response to Mycobacterium tuberculosis culture filtrate antigens in pulmonary tuberculosis

Association of HLA-DR2 genes/gene products has been shown with pulmonary tuberculosis (PTB) patients in India. In the present study, the influence of HLA-DR2 and non-DR2 genes/gene products on immunity to tuberculosis has been studied. Plasma samples of -DR2 positive patients (active and inactive TB) showed a higher antibody titre to Mycobacterium tuberculosis culture filtrate antigens than non-DR2 (-DR2 negative) patients. Immunoblot analysis revealed a trend towards an increased percentage of DR2 positive patients recognizing 38, 32/34 and 30/31 kDa antigens of M. tuberculosis than DR2 negative patients. A low spontaneous lymphoproliferative response (without antigen stimulation) was seen in HLA-DR2 positive active TB patients than HLA-DR2 negative patients. However, the antigen stimulated lymphocyte response was higher in the -DR2 positive patients (active and inactive TB) when compared to non-DR2 patients. Further, an inversional correlation between antibody titre and spontaneous as well as antigen induced lymphocyte response (measured by 3H thymidine uptake and expressed as counts per minute) was seen in HLA-DR2 positive active PTB patients than non-DR2 patients. The present study suggests that HLA-DR2 genes/gene products may be associated with a regulatory role in the mechanism of disease susceptibility to tuberculosis. The genes while augmenting the humoral immune response, they suppress the spontaneous and antigen induced lymphocyte response in -DR2 positive patients with active disease. Key words Antigen recognition - HLA-DR2-antibody titre - lymphocyte response - Mycobacteriu

HLA-DR phenotypes and IgG, IgA and IgM antibody responses to Mycobacterium tuberculosis culture filtrate and 30 kDa antigens in pulmonary tuberculosis

The role of HLA-DR genetic make-up on the IgG, IgA and IgM antibody response to Mycobacterium tuberculosis culture filtrate and 30 kDa antigens was studied in pulmonary tuberculosis. The study was carried out in HLA-DR typed active pulmonary tuberculosis (ATB) patients (n = 37), inactive (cured) pulmonary tuberculosis (ITB) patients (n = 79) and normal healthy subjects (NHS; n = 46). In ATB and ITB (cured) patients, IgG antibody (optical density at 490 nm for 1 : 3200 dilution) as measured by enzyme-linked immunosorbent assay was the predominant one than IgA and IgM antibodies. Increased IgG antibody titre to culture filtrate (P = 0.03) and decreased titre to 30 kDa antigen were observed with HLA-DR1-positive ATB patients than non-DR1 (ATB) patients. Moreover, HLA-DR4- and HLA-DR6-positive ATB patients showed trends toward an increased IgG antibody response to 30 kDa antigen than HLA-DR4- and HLADR6- negative (ATB) patients respectively. Significantly increased IgA antibody to 30 kDa antigen was observed with HLA-DR1-positive ATB patients than non-DR1 patients (P = 0.03). The study suggests that multiple HLA-DR molecules may regulate the IgG and IgA antibody responses to various proteins of M. tuberculosis. Moreover, HLA-DR phenotypes and increased IgG and IgA antibody titres may be useful to differentiate M. tuberculosis-infected subjects from normal subjects and cured patients with the same HLA-DR phenotypes or genetic make-up

Lymphocytotoxic antibodies & immunity in pulmonary tuberculosis

To understand whether the presence of cold reactive lymphocytotoxic antibodies (LCA) (reactive at 15°C) in the system has any effect on immunity to tuberculosis lymphocytotoxic antibodies to adherent cells (enriched-B ceils) and non-adherent cells were studied in active-TB (n=42) and inactive-TB (cured) patients (n=49) and healthy controls (n=32). The plasma samples of inactive-TB patients showed higher percentage of positivity for lymphocytotoxic antibodies (36.7%) than the active-TB patients (21.4%) and control subjects (18.8%). No significant difference on antibody and lymphocyte response against Mycobacterium tuberculosis culture filtrate antigens was observed between LCA positive and LCA negative active-TB patients and normal healthy controls. Further, determinationof HLA-DR phenotype of the patients and control subjects showed that individuals positive for lymphocytotoxic antibodies were more among HLA-DR2 positive and DR7 positive active-TB patients and control subjects than non-DR2 and non-DR7 subjects. The present study suggests that the cold reactive lymphocytotoxic antibodies may be against B-lymphocytes and persistent for a longer time. HLA-DR2 and -DR7 may be associated with the occurrence of LCA activity. Further, the presence of LCA has no immunoregulatory role on immunity to tuberculosis

Antibody and lymphocyte responses to Mycobacterium tuberculosis culture filtrate antigens in active and quiescent (cured) pulmonary tuberculosis.

Humoral and lymphocyte responses to Mycobacterium tuberculosis culture filtrate antigens were studied in active pulmonary tuberculosis (ATB) cases (n = 62), inactive (cured/quiescent) tuberculosis (ITB) patients (n = 62) and healthy control subjects (n = 60). Active tuberculosis patients showed very high antibody titre to M. tuberculosis culture filtrate antigens as compared to ITB and control subjects. M. tuberculosis antigens from 17 to 80 kDa were recognised by the plasma of all ATB and ITB patients as well as control subjects. However, the 38, 32-34, 30-31 and 27 kDa antigens were recognised more by the ATB patients as compared to the control subjects while the 64/66 kDa antigen was mostly recognised by the cured patients. Increased lymphocyte responses were seen with increasing concentrations of M. tuberculosis culture filtrate antigens and mitogens such as Phytohaemagglutinin (PHA) and Concanavalin-A (Con-A) in ATB and ITB patients as well as healthy control subjects. However, a low or suppressed lymphocyte response to PHA, Con-A and M. tuberculosis culture filtrate antigens was seen in ATB patients compared to ITB patients and control subjects. The study suggests that during the active stage of the disease, the humoral immune response is augmented but the antigen and mitogen induced lymphocyte response (an in vitro correlate of CMI response) is suppressed. This further suggests that the humoral immune response regulates the CMI response during the active stage of the disease; when the disease is cured, the antibody response declines and the lymphocyte response to antigens and mitogens increases to the same level as found in controls. This suggests that normal immune status gets restored in cured patients

HLA antigen profile in pulmonary tuberculosis patients and their spouses

HLA-A, -B, -DR and -DQ antigen profile was studied in pulmonary tuberculosis patients (n=209) and their spouses (family contacts; n=50) and healthy volunteers (n=72). An increased frequency of HLAA- 10, B7, B15, DR2 and DQ1 was seen in the pulmonary-TB (PTB) patients when compared to the total control subjects (n=122). However, a significant increase was seen only with HLA-DR2 (P < 0.001; Pc < 0.01; Relative Risk 2.3) and -DQ1 (P < 0.005; Pc<0.015; Relative Risk 2.8). Among the spouses and the corresponding patients, a similar increase of HLA-DR2 was seen. A decreased frequency of HLA-A19, B8, B17, B35, DR5 and DR6 were seen in PTB as compared to control groups. The present study suggested that HLA-DR2 and DQ1 genes/gene products may be associated with the susceptibility to tuberculosis either alone or in combination with other HLA or non-HLA genes

Association of HLA-Class I antigens and haplotypes with relapse of pulmonary tuberculosis in patients treated with short course chemotherapy

Whether or not there is an association between HLA antigen(s) and/or haplotypes aud relapse in patients successfully treated for pulmonary tuberculosis was examined. Serological determination of HLA -A, - B, -DR and -DQ antigens was carried out in patients with quiescent pulmonary tuberculosis and bacteriologically relapsed patients, after treatment with short course chemotherapy with Rifampicin, Isoniazid, Pyrazinamide and Streptomycin or Ethambutol in various combinations for 6-8 month Au increased antigen frequency of HLA -Al (P = 0.03) and B17 was seen in patients with bacteriological relapse compared with those with quiescent disease. The relative risks (RR) were Al = 2.8 and B17 = 3.2, respectively, The haplotypes Al-B17 (RR = 3.3), B17-DR7 (RR = 3.0), Al-DR7 (p = 0.04; RR = 9.3) were very common in patients with bacteriological relapse. This increase of HLA-Al, B17 antigens or the haplotypes Al- B 17, B17-DR7 or Al-DR7 (P = 0.04) was seen irrespective of the treatment regimen. The present study suggests that HLA -Al (and - B17) antigen(s), as such, and/or haplotypes Al- : DR7 or non-HLA genes linked closer to HLA -A, -B and -DR loci may be associated with relapse of pulmonary tuberculosis, after chemotherapy

A Pilot Study Identifying Brain-Targeting Adaptive Immunity in Pediatric Extracorporeal Membrane Oxygenation Patients with Acquired Brain Injury

OBJECTIVES: Extracorporeal membrane oxygenation provides short-term cardiopulmonary life support, but is associated with peripheral innate inflammation, disruptions in cerebral autoregulation, and acquired brain injury. We tested the hypothesis that extracorporeal membrane oxygenation also induces CNS-directed adaptive immune responses which may exacerbate extracorporeal membrane oxygenation-associated brain injury. DESIGN: A single center prospective observational study. SETTING: Pediatric and cardiac ICUs at a single tertiary care, academic center. PATIENTS: Twenty pediatric extracorporeal membrane oxygenation patients (0-14 yr; 13 females, 7 males) and five nonextracorporeal membrane oxygenation Pediatric Logistic Organ Dysfunction score matched patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Venous blood samples were collected from the extracorporeal membrane oxygenation circuit at day 1 (10-23 hr), day 3, and day 7 of extracorporeal membrane oxygenation. Flow cytometry quantified circulating innate and adaptive immune cells, and CNS-directed autoreactivity was detected using an in vitro recall response assay. Disruption of cerebral autoregulation was determined using continuous bedside near-infrared spectroscopy and acquired brain injury confirmed by MRI. Extracorporeal membrane oxygenation patients with acquired brain injury (n = 9) presented with a 10-fold increase in interleukin-8 over extracorporeal membrane oxygenation patients without brain injury (p \u3c 0.01). Furthermore, brain injury within extracorporeal membrane oxygenation patients potentiated an inflammatory phenotype in adaptive immune cells and selective autoreactivity to brain peptides in circulating B cell and cytotoxic T cell populations. Correlation analysis revealed a significant relationship between adaptive immune responses of extracorporeal membrane oxygenation patients with acquired brain injury and loss of cerebral autoregulation. CONCLUSIONS: We show that pediatric extracorporeal membrane oxygenation patients with acquired brain injury exhibit an induction of pro-inflammatory cell signaling, a robust activation of adaptive immune cells, and CNS-targeting adaptive immune responses. As these patients experience developmental delays for years after extracorporeal membrane oxygenation, it is critical to identify and characterize adaptive immune cell mechanisms that target the developing CNS

Visualization and Quantification of Post-Stroke Neural Connectivity and Neuroinflammation Using Serial Two-Photon Tomography in the Whole Mouse Brain

Whole-brain volumetric microscopy techniques such as serial two-photon tomography (STPT) can provide detailed information on the roles of neuroinflammation and neuroplasticity throughout the whole brain post-stroke. STPT automatically generates high-resolution images of coronal sections of the entire mouse brain that can be readily visualized in three dimensions. We developed a pipeline for whole brain image analysis that includes supervised machine learning (pixel-wise random forest models via the “ilastik” software package) followed by registration to a standardized 3-D atlas of the adult mouse brain (Common Coordinate Framework v3.0; Allen Institute for Brain Science). These procedures allow the detection of cellular fluorescent signals throughout the brain in an unbiased manner. To illustrate our imaging techniques and automated image quantification, we examined long-term post-stroke motor circuit connectivity in mice that received a motor cortex photothrombotic stroke. Two weeks post-stroke, mice received intramuscular injections of pseudorabies virus (PRV-152), a trans-synaptic retrograde herpes virus driving expression of green fluorescent protein (GFP), into the affected contralesional forelimb to label neurons in descending tracts to the forelimb musculature. Mice were sacrificed 3 weeks post-stroke. We also quantified sub-acute neuroinflammation in the post-stroke brain in a separate cohort of mice following a 60 min transient middle cerebral artery occlusion (tMCAo). Naive e450+-labeled splenic CD8+ cytotoxic T cells were intravenously injected at 7, 24, 48, and 72 h post-tMCAo. Mice were sacrificed 4 days after stroke. Detailed quantification of post-stroke neural connectivity and neuroinflammation indicates a role for remote brain regions in stroke pathology and recovery. The workflow described herein, incorporating STPT and automated quantification of fluorescently labeled features of interest, provides a framework by which one can objectively evaluate labeled neuronal or lymphocyte populations in healthy and injured brains. The results provide region-specific quantification of neural connectivity and neuroinflammation, which could be a critical tool for investigating mechanisms of not only stroke recovery, but also a wide variety of brain injuries or diseases